A novel air-dried multiplex high-resolution melt assay for the detection of extended-spectrum β-lactamase and carbapenemase genes

Abstract

Objectives: This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended-spectrum β-lactamase (ESBL) (bla_SHV and bla_CTX-M groups 1 and 9) and carbapenemase (bla_NDM, bla_IMP, bla_KPC, bla_VIM and bla_OXA-48-like) genes that confer resistance to cephalosporins and carbapenems.

Methods: The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and the UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility. Assay reproducibility was evaluated across five different real-time quantitative PCR (qPCR) instruments [Rotor-Gene® Q, QuantStudio^TM 5, CFX96, LightCycler® 480 and Magnetic Induction Cycler (Mic)]. Assay stability was also assessed under different storage temperatures (6.2 ± 0.9°C, 20.4 ± 0.7°C and 29.7 ± 1.4°C) at six time points over 8 months.

Results: The sensitivity and specificity (with 95% confidence intervals) for detecting ESBL and carbapenemase genes was 94.7% (92.5-96.5%) and 99.2% (98.8-99.5%) compared with the reference gel-based PCR and sequencing and 98.3% (97.0-99.3%) and 98.5% (98.0-98.9%) compared with the original HRM wet PCR mix format. Overall agreement was 91.1% (90.0-92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay, and no loss of sensitivity occurred under all storage conditions and time points.

Conclusion: We present a ready-to-use air-dried HRM PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve antimicrobial resistance detection.

Keywords: Antimicrobial resistance; Carbapenemase; ESBL; Extended-spectrum β-lactamase; High-resolution melting; Molecular diagnostics.

Fig. 1

Melt curve profile of the air-dried high-resolution melt (HRM) assay showing (a) the panel comprising the eight markers, with some of the isolates also being co-producers and (b) detail of the simultaneous detection of two (pink), three (green) and four (blue) genes in isolates co-producing extended-spectrum β-lactamases (ESBLs) and carbapenemases. (a) Orange, Klebsiella pneumoniae harbouring bla_SHV and bla_IMP genes; red, Escherichia coli harbouring bla_OXA-48-like and bla_CTX-M-9 genes; pink, K. pneumoniae harbouring bla_SHV and bla_KPC genes; blue, K. pneumoniae harbouring bla_SHV and bla_VIM genes; purple, Acinetobacter spp. harbouring the bla_NDM gene; and grey, Pseudomonas aeruginosa harbouring the bla_IMP gene. (b) Pink, K. pneumoniae harbouring bla_SHV and bla_CTX-M-1 genes; green, K. pneumoniae harbouring bla_OXA-48-like, bla_SHV and bla_CTX-M-1 genes; and blue, K. pneumoniae harbouring bla_OXA-48-like, bla_NDM, bla_SHV and bla_CTX-M-1 genes.

Fig. 2

Melting temperatures (T_m) of the eight amplicons of the air-dried high-resolution melt (HRM) assay run in the CFX96, QuantStudio^TM 5 (QSTUDIO), Rotor-Gene® Q (RotorGene-Q), LightCycler® 480 (LC48) and Magnetic Induction Cycler (Mic). The whiskers show the maximum and minimum values, with the exception of outliers (circles) and extremes (rhombus).

Fig. 3

Plate mean fluoresce peak height at the beginning of study (T0) and after 1 week (T1), 2 weeks (T2), 1 month (T3), 3 months (T4) and 8 months (T5) under fridge storage (6.2 ± 0.9°C), at room temperature (20.4 ± 0.7°C) and in an oven (29.7 ± 1.4°C). The colour of asterisks indicates which storage conditions were statistically different between time points: blue (fridge), orange (room temperature), red (oven) and black (all temperature conditions). CI, confidence interval.

Fig. 4

Peak height of isolate 1 (bla_CTX-M-1-positive), isolate 2 (bla_SHV-positive) and isolate 3 (bla_CTX-M-1- and bla_SHV-positive) at the limit of detection (LOD) dilution at different timepoints and storage conditions.